Involvement of Akt/NF‐κB pathway in N6‐isopentenyladenosine‐induced apoptosis in human breast cancer cells

N⁶‐isopentenyladenosine (i6A) inhibits the tumor cell growth by inducing cell apoptosis in various cancer cell lines. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. In this study, we further explored the molecular mechanisms of i6A as an anticancer agent on a human breast cancer cell line MDA MB 231. Treatment with i6A decreased the cell proliferation of MDA MB 231 cells in a dose‐dependent manner by arresting the cells at G₀/G₁ phase. This effect was strongly associated with concomitant decrease in the level of cyclin D1, cyclin E, cdk2, and increase of p21waf1 and p27kip. In addition i6A also induced apoptotic cell death by increasing the expression of Bax, and decreasing the levels of Bcl‐2 and Bcl‐xL, and subsequently triggered mitochondria apoptotic pathway (release of cytochrome c and activation of caspase‐3). We observed that i6A suppressed the nuclear factor kappaB (NF‐κB) pathway and inhibited the Akt activation. The results of this study indicate that i6A decreases cell proliferation and induces apoptotic cell death in human breast cancer cells, possibly by decreasing signal transduction through the Akt/NF‐κB cell survival pathway. © 2010 Wiley‐Liss, Inc.     

The antitumor activity of the fungicide ciclopirox

Ciclopirox olamine (CPX) is a synthetic antifungal agent clinically used to treat mycoses of the skin and nails. Here, we show that CPX inhibited tumor growth in human breast cancer MDA‐MB‐231 xenografts. To unveil the underlying mechanism, we further studied the antitumor activity of CPX in cell culture. The results indicate that CPX inhibited cell proliferation and induced apoptosis in human rhabdomyosarcoma (Rh30), breast carcinoma (MDA‐MB231) and colon adenocarcinoma (HT‐29) cells in a concentration‐dependent manner. By cell cycle analysis, CPX induced accumulation of cells in G₁/G₀ phase of the cell cycle. Concurrently, CPX downregulated cellular protein expression of cyclins (A, B1, D1 and E) and cyclin‐dependent kinases (CDK2 and CDK4) and upregulated expression of the CDK inhibitor p21^Cip1, leading to hypophosphorylation of retinoblastoma protein. CPX also downregulated protein expression of Bcl‐xL and survivin and enhanced cleavages of Bcl‐2. Z‐VAD‐FMK, a pan‐caspase inhibitor, partially prevented CPX‐induced cell death, suggesting that CPX‐induced apoptosis of cancer cells is mediated at least in part through caspase‐dependent mechanism. The results indicate that CPX is a potential antitumor agent.     

5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone‐induced cell death in human Leukemia cells is dependent on caspases and activates the MAPK pathway

Flavonoids are polyphenolic compounds which display a vast array of biological activities and are promising anticancer agents. In this study we investigated the effect of 5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone (THDF) on viability of nine human tumor cell lines and found that it was highly cytotoxic against leukemia cells. THDF induced G₂–M phase cell‐cycle arrest and apoptosis through a caspase‐dependent mechanism involving cytochrome c release, processing of multiple caspases (caspase‐3, ‐6, ‐7, and ‐9) and cleavage of poly(ADP‐ribose) polymerase. Overexpression of the protective mitochondrial proteins Bcl‐2 and Bcl‐x_L conferred partial resistance to THDF‐induced apoptosis. This flavonoid induced the phosphorylation of members of the mitogen‐activated protein kinases (MAPKs) family and cell death was attenuated by inhibition of c‐jun N‐terminal kinases/stress‐activated protein kinases (JNK/SAPK) and of extracellular signal‐regulated kinases (ERK) 1/2. In the present study we report that THDF‐induced cell death is mediated by an intrinsic dependent apoptotic event involving mitochondria and MAPKs, and through a mechanism independent of the generation of reactive oxygen species. The results suggest that THDF could be useful in the development of novel anticancer agents. Mol. Carcinog. © 2010 Wiley‐Liss, Inc.     

Naturally occurring asteriscunolide A induces apoptosis and activation of mitogen‐activated protein kinase pathway in human tumor cell lines

Sesquiterpene lactones have attracted much attention because they display a wide range of biological activities, including antitumor properties. Here, we show the effects of the naturally occurring sesquiterpene lactone asteriscunolide A (AS) on viability of human melanoma, leukemia and cells that overexpress antiapoptotic proteins, namely Bcl‐2 and Bcl‐x_L. All cell lines were sensitive to this compound, with IC₅₀ values of ～5 µM. The cytotoxic effects of AS were accompanied by a G₂‐M phase arrest of the cell cycle and a concentration‐ and time‐dependent appearance of apoptosis as determined by DNA fragmentation, translocation of phosphatidylserine to the cell surface and sub‐G₁ ratio. Apoptosis was associated with caspase‐3 activity and poly(ADP‐ribose) polymerase cleavage and was prevented by the nonspecific caspase inhibitor z‐VAD‐fmk, indicating that caspases are essential components in this pathway. The apoptotic effect of AS was also associated with (i) the release of cytochrome c from mitochondria which was accompanied by dissipation of the mitochondrial membrane potential (ΔΨ_m) and (ii) the activation of the mitogen‐activated protein kinases (MAPKs) pathway. AS‐induced cell death was potentiated by inhibition of extracellular signal‐regulated kinases (ERK) 1/2 signaling with U0126 and PD98059. Intracellular reactive oxygen species (ROS) seem to play a pivotal role in this process since high levels of ROS were produced early (1 h) and apoptosis was completely blocked by the free radical scavenger N‐acetyl‐L ‐cysteine (NAC). The present study demonstrates that AS‐induced cell death is mediated by an intrinsic‐dependent apoptotic event involving mitochondria and MAPKs, and through a mechanism dependent on ROS generation. © 2010 Wiley‐Liss, Inc.     

Resveratrol induces apoptosis and cell cycle arrest of human T24 bladder cancer cells in vitro and inhibits tumor growth in vivo

Resveratrol, a naturally occurring polyphenolic antioxidant compound present in grapes and red wine, has been reported to hold various biochemical responses. In this preliminary study, we evaluate the chemopreventive potential of resveratrol against bladder cancer and its mechanism of action. Treatment of bladder cancer cells with resveratrol resulted in a significant decrease in cell viability. Resveratrol induced apoptosis through the modulation of Bcl‐2 family proteins and activation of caspase 9 and caspase 3 followed by poly(ADP‐ribose) polymerase degradation. Treatment with resveratrol led to G₁ phase cell cycle arrest in T24 cells by activation of p21 and downregulation of cyclin D1, cyclin‐dependent kinase 4, and phosphorylated Rb. Resveratrol also inhibited the phosphorylation of Akt, whereas the phosphorylation of p38 MAPK was enhanced. In addition, resveratrol treatment decreased the expression of vascular endothelial growth factor and fibroblast growth factor‐2, which might contribute to the inhibition of tumor growth on the bladder cancer xenograft model. These findings suggest that reveratrol could be an important chemoprevention agent for bladder cancer. (Cancer Sci 2009)     

Co-treatment with ginsenoside Rh2 and betulinic acid synergistically induces apoptosis in human cancer cells in association with enhanced capsase-8 activation, bax translocation, and cytochrome c release

We provide evidence for the first time, that two natural compounds ginsenoside Rh2 (G‐Rh2) and betulinic acid (Bet A) synergistically induce apoptosis in human cervical adenocarcinoma (HeLa), human lung cancer A549, and human hepatoma HepG2 cells. G‐Rh2 and Bet A cooperated to induce Bax traslocation to mitochondria and cytochrome c release. Co‐treatment of G‐Rh2 and Bet A resulted in enhanced cleavage of caspase‐8 and Bid. Moreover, specific inhibition of caspase‐8 by siRNA technology effectively reduced caspase‐9 processing, poly (ADP‐ribose) polymerase (PARP) cleavage, caspase‐3 activation, and apoptosis in co‐treated cells, which indicated that the caspase‐8 feedback amplification pathway may have been involved in the apoptosis process. A previous study has shown that G‐Rh2 induces cancer cell apoptosis via a Bcl‐2 and/or Bcl‐xL‐independent mechanism, and Bet A induces apoptosis mainly through a mitochondrial pathway with tumor specificity. Since the antiapoptotic Bcl‐2 and Bcl‐xL are frequently overexpressed in human cancer cells, combined treatment with G‐Rh2 and Bet A may be a novel strategy to enhance efficacy of anticancer therapy. © 2011 Wiley‐Liss, Inc.     

Apoptosis induced by methylene‐blue‐mediated photodynamic therapy in melanomas and the involvement of mitochondrial dysfunction revealed by proteomics

Methylene blue (MB) is a widely studied agent currently under investigation for its properties relating to photodynamic therapy (PDT). Recent studies have demonstrated that MB exhibits profound phototoxicity affecting a variety of tumor cell lines. However, the mechanistic explanation for methylene‐blue‐mediated photodynamic therapy (MB‐PDT) in the context of melanoma therapy is still obscure. In the present study, B16F1 melanoma cells were treated by MB‐PDT under different conditions, and thereafter subjected to cell viability detection assays. MB‐PDT could induce intense apoptotic cell death through a series of steps beginning with the photochemical generation of reactive oxygen species that activate the caspase‐9/caspase‐3 apoptosis pathway. Blocking activation of caspase‐3 and induction of oxidative stress by caspase inhibitor and by glutathione, respectively, markedly reduced apoptotic cell death in vitro. Importantly, proteomics study defining altered protein expression in treated cells suggests the involvement of several mitochondrial proteins playing important roles in electron transfer chain, implying mitochondrial dysfunction during the treatment. Furthermore, a transplantable mouse melanoma model was utilized to estimate the effectiveness of MB‐PDT in vivo. The treated mice displayed decreased tumor size and prolonged survival days, which was associated with enhanced apoptotic cell death. These results, offering solid evidence of the induction of mitochondria‐related apoptosis in tumor cells, reveal new aspects of MB‐PDT having potential to be a palliative treatment of melanoma. (Cancer Sci 2008; 99: 2019–2027)     

Antitumor effects of novel compound,guttiferone K,on colon cancer by p21Waf1/Cip1‐mediated G0/G1 cell cycle arrest and apoptosis

Low selectivity is one of the major problems of currently used anticancer drugs, therefore, there is a high demand for novel, selective antitumor agents. In this study, the anticancer effects and mechanisms of guttiferone K (GUTK), a novel polyprenylated acylphloroglucinol derivative isolated from Garcinia cowa Roxb., were examined for its development as a novel drug targeting colon cancer. GUTK concentration‐ and time‐dependently reduced the viability of human colon cancer HT‐29 cells (IC₅₀ value 5.39 ± 0.22 μM) without affecting the viability of normal human colon epithelial CCD 841 CoN cells and induced G₀/G₁ cell cycle arrest in HT‐29 cells by down‐regulating cyclins D1, D3 and cyclin‐dependent kinases 4 and 6, while selectively restoring p21Waf1/Cip1 and p27Kip1 to levels comparable to those observed in normal colon cells, without affecting their levels in normal cells. GUTK (10.0 μM) induced cleavage of PARP, caspases‐3, ‐8 and ‐9 and chromatin condensation to stimulate caspase‐3‐mediated apoptosis. The addition of a JNK inhibitor, SP600125, partially reversed GUTK‐induced caspase‐3 activity, indicating the possible involvement of JNK in GUTK‐induced apoptosis. Furthermore, GUTK (10 mg/kg, i.p.) significantly decreased the tumor volume in a syngeneic colon tumor model when used alone or in combination with 5‐fluorouracil without toxicity to the mice. Immunohistochemical staining of the tumor sections revealed a mechanism involving an increase in cleaved caspase‐3 and a decrease in cell proliferation marker Ki‐67. Our results support GUTK as a promising novel, potent and selective antitumor drug candidate for colon cancer.     

Targeting CDK9 by wogonin and related natural flavones potentiates the anti‐cancer efficacy of the Bcl‐2 family inhibitor ABT‐263

Tumor initiation, progression and resistance to therapies are tightly associated with over‐expression of anti‐apoptotic proteins Bcl‐2, Bcl‐x_L, Bcl‐w and Mcl‐1. ABT‐263 (Navitoclax), an orally bio‐available small‐molecule mimetic of the Bcl‐2 homology domain 3, inhibits Bcl‐2, Bcl‐x_L, and Bcl‐w and has shown anti‐cancer effects mainly on lymphomas and lymphocytic leukemia. Despite promising results obtained from the clinical trials, the use of ABT‐263 in patients is dose‐limited due to causing thrombocytopenia via inhibition of Bcl‐x_L in platelets. ABT‐199 specifically inhibits Bcl‐2; however, its use is limited to tumors over‐expressing only Bcl‐2. Besides, many tumors resist treatment due to high levels of Mcl‐1 expression or develop resistance via up‐regulation of Mcl‐1 during long‐term exposure. These obstacles highlight the demand to improve the ABT‐263‐based therapy. In this study, we show that anti‐cancer flavones, e.g., wogonin, baicalein, apigenin, chrysin and luteolin enhance ABT‐263‐induced apoptosis in different cancer cell lines and in primary AML and ALL cells by down‐regulation of Mcl‐1 expression. Importantly, wogonin does not enhance the toxicity of ABT‐263 to proliferating normal T cells and thrombocytes. Wogonin also potentiates the lethality of ABT‐263 in cancer cells which have acquired resistance to ABT‐263. Furthermore, we show that combination of wogonin with ABT‐263 promotes in vivo tumor regression in a human T‐cell leukemia xenograft mouse model. Our study demonstrates that wogonin (and related flavones) reduce the effective dose of ABT‐263 thereby possibly decreasing the risk of adverse side effects.     

The dual PI3K/mTOR inhibitor PI‐103 promotes immunosuppression,in vivo tumor growth and increases survival of sorafenib‐treated melanoma cells

Melanoma is the most lethal human skin cancer. If metastatic, it becomes very aggressive and resistant to standard modalities of anticancer treatment. During the last 10 years, several therapeutic strategies have been tested including the use of single and combined small drugs. Experimental results indicate that RAS and PI3K pathways are important for the development and maintenance of melanoma. In this study, we assessed the in vitro and in vivo inhibition potential of PI‐103, a PI3K (p110α)/mTOR inhibitor and sorafenib, a BRAF inhibitor, as single agents and in combination in primary melanoma cell lines. Although PI‐103 and sorafenib inhibited melanoma in vitro cell proliferation and viability, the inhibition of RAS pathway appeared to be more effective. The combination of the two agents in in vitro showed a synergistic effect inhibiting RAS and PI3K pathways in a cell line dependent manner. However, no cooperative effect was observed in blocking in vivo tumor growth in immunocompetent mice. In contrary to the expected, the data indicate that PI‐103 induced immunosuppression promoting in vivo tumor growth and inhibiting apoptosis. Furthermore, in vitro studies examining the effects of the PI3K/mTOR inhibitor in tumor derived cell lines indicated that PI‐103 induced the anti‐apoptotic BH3 family proteins Mcl1, Bcl2 and Bcl_xL favoring, the in vitro survival of sorafenib treated melanoma cells. These data certainly makes an argument for investigating unexpected effects of rational drug combinations on immunocompetent animal models prior to conducting clinical studies.     

Lysosomal destabilization and cathepsin B contributes for cytochrome c release and caspase activation in embelin‐induced apoptosis

XIAP is an important antiapoptotic protein capable of conferring resistance to cancer cells. Embelin, the small molecular inhibitor of XIAP, possesses wide spectrum of biological activities with strong inhibition of nuclear factor kappa B and downstream antiapoptotic genes. However, the mechanism of its cell death induction is not known. Our studies using colon cancer cells lacking p53 and Bax suggest that both lysosomes and mitochondria are prominent targets of embelin‐induced cell death. Embelin induced cell‐cycle arrest in G₁ phase through p21, downstream of p53. In the absence of p21, the cells are sensitized to death in a Bax‐dependent manner. The loss of mitochondrial membrane potential induced by embelin was independent of Bax and p53, but lysosomal integrity loss was strongly influenced by the presence of p53 but not by Bax. Lysosomal role was further substantiated by enhanced cathepsin B activity noticed in embelin‐treated cells. p53‐dependent lysosomal destabilization and cathepsin B activation contribute for increased sensitivity of p21‐deficient cells to embelin with enhanced caspase 9 and caspase 3 activation. Cathepsin B inhibitor reduced cell death and cytochrome c release in embelin‐treated cells indicating lysosomal pathway as the upstream of mitochondrial death signaling. Deficiency of cell‐cycle arrest machinery renders cells more sensitive to embelin with enhanced lysosomal destabilization and caspase processing emphasizing its potential therapeutic importance to address clinical drug resistance. © 2009 Wiley‐Liss, Inc.     

The marine triterpene glycoside frondoside A exhibits activity in vitro and in vivo in prostate cancer

Despite recent advances in the treatment of metastatic castration‐resistant prostate cancer (CRPC), outcome of patients remains poor due to the development of drug resistance. Thus, new drugs are urgently needed. We investigated efficacy, toxicity and mechanism of action of marine triterpene glycoside frondoside A (FrA) using CRPC cell lines in vitro and in vivo. FrA revealed high efficacy in human prostate cancer cells, while non‐malignant cells were less sensitive. Remarkably, proliferation and colony formation of cells resistant to enzalutamide and abiraterone (due to the androgen receptor splice variant AR‐V7) were also significantly inhibited by FrA. The marine compound caused cell type specific cell cycle arrest and induction of caspase‐dependent or ‐independent apoptosis. Up‐regulation or induction of several pro‐apoptotic proteins (Bax, Bad, PTEN), cleavage of PARP and caspase‐3 and down‐regulation of anti‐apoptotic proteins (survivin and Bcl‐2) were detected in treated cells. Global proteome analysis revealed regulation of proteins involved in formation of metastases, tumor cell invasion, and apoptosis, like keratin 81, CrkII, IL‐1β and cathepsin B. Inhibition of pro‐survival autophagy was observed following FrA exposure. In vivo, FrA inhibited tumor growth of PC‐3 and DU145 cells with a notable reduction of lung metastasis, as well as circulating tumor cells in the peripheral blood. Increased lymphocyte counts of treated animals might indicate an immune modulating effect of FrA. In conclusion, our results suggest that FrA is a promising new drug for the treatment of mCRPC. Induction of apoptosis, inhibition of pro‐survival autophagy, and immune modulatory effects are suspected modes of actions.     

2-Methoxyestradiol and multidrug resistance: can 2-methoxyestradiol chemosensitize resistant breast cancer cells?

2-Methoxyestradiol (2ME), a natural derivative of estradiol, is currently evaluated in clinical trials for breast cancer. The current study aims to evaluate the modulatory effects of 2ME on regulation of multidrug resistance (MDR) in doxorubicin (Dox) resistant breast cancer cells (MCF-7/Dox) and its underlying mechanisms. The chemosensitizing effect of 2ME on Dox cytotoxicity is tested by MTT assay. RT² Profiler PCR Array was used to identify differentially expressed genes in Dox and/or 2ME treatment groups, based on significance of results 4 genes were selected: MDR1, Bcl2, P53 and Cyclin D1. The expression of these genes was confirmed using western blotting. Lastly, functions of these genes were examined by studying p-glycoprotein (p-gp) function, caspase 3 activity and flowcytometric cell cycle assays respectively. 2ME significantly increased sensitivity of the resistant MCF-7/Dox cells to the cytotoxic effect of Dox by 2.9-folds. The array and western blotting showed that Bcl2 and Cyclin D1 expression were down regulated; P53 expression was not affected while MDR1 was over expressed by combination of 2ME with Dox. 2ME increased p-gp function by 24 ± 7.05%, compared to control. Addition of 2ME to Dox increased caspase activity by 27-folds. Combination of 2ME to Dox arrested the cell cycle in G₁ and S phases, compared to Dox. In conclusion, 2ME chemosensitizes resistant breast cancer cells to Dox cytotoxcity by down regulating expression of Bcl2 and Cyclin D1, augmenting caspase 3 activity as well as inducing cell cycle block in G₁ and S phases.     

Identification of predictive factors of response to the BH3‐mimetic molecule ABT‐737: An ex vivo experiment in human serous ovarian carcinoma

Ovarian cancers are addicted to Bcl‐x_L and Mcl‐1, antiapoptotic members of the Bcl‐2 family. Bcl‐x_L can be inhibited by the BH3‐mimetic ABT‐737. In vitro, ABT‐737 can induce apoptosis of cancer cells, and its activity is potentiated by Mcl‐1 inactivation. Herein, we assessed the sensitivity of human ovarian tumor nodes to ABT‐737 when combined with carboplatin, which can indirectly inhibit Mcl‐1. Fresh samples from 25 patients with high‐grade serous ovarian cancer (HGSOC) who were chemo‐naïve and had undergone surgery were prospectively exposed ex vivo to ABT‐737 ± carboplatin. The treatment effect was studied on sliced tumor nodes by assessment of cleaved‐caspase 3 immunostaining. We also studied the association between baseline Bcl‐2 family protein expression (via immunohistochemistry) and the response of nodes to treatment. ABT‐737 induced apoptosis as a single agent but its efficacy was not improved by the addition of carboplatin. Bim was frequently expressed (20/25) and its absence or low expression was associated with the absence of response to ABT‐737, p value = 0.019 by Fisher's test and sensitivity = 93%, (95% confidence interval, 66–100). Moreover, we observed that in tumors in which Bim was expressed, a low expression of phospho‐Erk1/2 or Mcl‐1 improved the proportion of responses. This pilot study showed that ABT‐737 has promise as monotherapy for HGSOC in a specific subgroup of tumors. Bim, Mcl‐1, and phospho‐Erk1/2 appeared to be relevant biomarkers that could be used for the selection of patients in the design of clinical trials using Navitoclax (an orally available compound related to ABT‐737).     

Functional screening identifies a microRNA,miR‐491 that induces apoptosis by targeting Bcl‐XL in colorectal cancer cells

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate expression of target mRNA. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and considered as new therapeutic targets for cancers. In our study, we performed a gain‐of‐function screen using 319 miRNAs to identify those affecting cell proliferation and death in human colorectal cancer cells (DLD‐1). We discovered a number of miRNAs that increased or decreased cell viability in DLD‐1. They included known oncogenic miRNAs such as miR‐372 and miR‐373, and tumor suppressive miRNAs such as miR‐124a, but also some for which this information was novel. Among them, miR‐491 markedly decreased cell viability by inducing apoptosis. We demonstrated that Bcl‐X_L was a direct target of miR‐491, and its silencing contributed to miR‐491‐induced apoptosis. Moreover, treatment of miR‐491 suppressed in vivo tumor growth of DLD‐1 in nude mice. Our study provides a new regulation of Bcl‐X_L by miR‐491 in colorectal cancer cells, and suggests a therapeutic potential of miRNAs for treating colorectal cancer by targeting Bcl‐X_L.     

The novel histone deacetylase inhibitor,AR‐42, inhibits gp130/Stat3 pathway and induces apoptosis and cell cycle arrest in multiple myeloma cells

Multiple myeloma (MM) remains incurable with current therapy, indicating the need for continued development of novel therapeutic agents. We evaluated the activity of a novel phenylbutyrate‐derived histone deacetylase inhibitor, AR‐42, in primary human myeloma cells and cell lines. AR‐42 was cytotoxic to MM cells at a mean LC₅₀ of 0.18 ± 0.06 μmol/l at 48 hr and induced apoptosis with cleavage of caspases 8, 9 and 3, with cell death largely prevented by caspase inhibition. AR‐42 downregulated the expression of gp130 and inhibited activation of STAT3, with minimal effects on the PI3K/Akt and MAPK pathways, indicating a predominant effect on the gp130/STAT‐3 pathway. AR‐42 also inhibited interleukin (IL)‐6‐induced STAT3 activation, which could not be overcome by exogenous IL‐6. AR‐42 also downregulated the expression of STAT3‐regulated targets, including Bcl‐xL and cyclin D1. Overexpression of Bcl‐xL by a lentivirus construct partly protected against cell death induced by AR‐42. The cyclin dependent kinase inhibitors, p16 and p21, were also significantly induced by AR‐42, which together with a decrease in cyclin D1, resulted in G₁ and G₂ cell cycle arrest. In conclusion, AR‐42 has potent cytotoxicity against MM cells mainly through gp130/STAT‐3 pathway. The results provide rationale for clinical investigation of AR‐42 in MM.